Pharmacokinetics of anti-infectious reagents in silkworms

Abstract

Silkworm microorganism infection models are useful for screening novel therapeutically effective antimicrobial agents. In this study, we used silkworms to investigate the pharmacokinetics and metabolism of antimicrobial agents, in which cytochrome P450 plays a major role. The pharmacokinetic parameters of the antimicrobial agents were determined based on their concentrations in the hemolymph after administration. The parameters, such as half-lives and distribution volumes, in silkworm were consistent with those in mammalian models. In addition, antifungal agents with reduced therapeutic effectiveness due to high protein-binding capacities in mammalian serum exhibited similar features in silkworm hemolymph. Cytochrome P450 enzymes, which metabolize exogenous compounds in mammalian liver, were distributed mainly in the silkworm midgut. Most of the compounds metabolized by cytochrome P450 in humans are also metabolized in the silkworm midgut. These findings suggest that the pharmacokinetics of antimicrobial agents are fundamentally similar between silkworms and mammals, and that therapeutic effects in the silkworm infection model reflect the pharmacokinetics of the test samples.

Figure 1

Time-course of the decrease in the concentration of antibiotics in silkworm hemolymph. Antibiotics were injected into silkworm hemolymph and hemolymph was collected by cutting the legs at the indicated time. Concentration of each antibiotic was determined by HPLC. (A) Chloramphenicol (100 µg/larva), (B) tetracycline (20 µg/larva), (C) vancomycin (50 µg/larva), (D) rifampicin (50 µg/larva), (E) micafungin (50 µg/larva), and (F) fluconazole (50 µg/larva).

Figure 2

Tissue specificity of 7-ethoxycoumarin deethylation activity in silkworm. (A) The midgut, fat body, silk gland, and Malpighian tubule were extirpated from silkworms and incubated in medium containing 7-ethoxycoumarin at 30 °C. Medium was collected, extracted by acetone, and evaporated. Samples discovered in 50 mM NaOAc buffer (pH 6.0) were treated with beta-glucosidase. The amount of 7-hydroxycomarin, a metabolite, was determined by fluorometry. (B) Inhibition of 7-ethoxycoumarin deethylation by cimetidine in the silkworm midgut. Silkworm midgut was incubated in medium containing 7-ethoxycoumarin and cimetidine at 30 °C. Samples were collected, extracted by acetone, and treated with beta-glucosidase. The amount of 7-hydroxycomarin was determined by fluorometry.

Figure 3

Decay of cytochrome P450 substrates in in vitro culture of the silkworm midgut. Silkworm midgut was incubated in medium with cytochrome P450 substrates at 30 °C. The samples were collected, extracted by acetone, and evaporated. The amounts of substrates were determined by HPLC analysis. (A) Omeprazole, (B) rifampicin, (C) ketoconazole, (D) diclofenac, (E) testosterone, and (F) caffeine.